Prevalence and Resistance Profiles of Pediatric Enterococcal Isolates: A Five-Year Update from Children's Medical Center Hospital, Tehran

Background In this study, attempts were made to evaluate the frequency of high-level gentamicin-resistant (HLGR) and vancomycin-resistant enterococci (VRE) and the prevalence and antibiotic resistance profile of enterococcal species isolated from pediatric patients referred to Children's Medical Center Hospital, Tehran, over five years. Materials and Methods A total of 404 enterococcal isolates from different patients referred to the Children's Medical Center between March 2016 and March 2021 were included in this cross-sectional study. Antimicrobial susceptibility testing was performed using standard methods according to the guidelines of the Clinical Laboratories Standards Institute (CLSI). Results Approximately one-third of the enterococcal strains were isolated from urology and intensive care units. 17.3% of the isolates were obtained from outpatient sources. However, 82.7% of the isolates were sourced from inpatient settings. We found that the rates of resistance to ampicillin, penicillin, and vancomycin were twice as high in inpatients as in outpatients. Of the total isolates, 87.4% and 49.3% were identified as HLGR and VRE, respectively. In addition, we identified 2% of the VRE isolates that were not susceptible to linezolid. Nitrofurantoin showed excellent activity against enterococcal isolates in the urine, with a susceptibility rate of 92.5%. Conclusion The present study reports the highest range of VRE isolated from pediatric patients in Iran. Despite the predominance of HLGR enterococci in our region, vancomycin remains effective against such strains. This study is among the few to demonstrate the incidence of linezolid-insensitive VRE in pediatric patients. Therefore, it is important to evaluate effective infection control measures to prevent linezolid and vancomycin resistance in enterococci.


Introduction
Enterococcus species, particularly Enterococcus faecalis and Enterococcus faecium, are common opportunistic bacteria found in the human gastrointestinal (GI) tract, vagina, and oral cavity.Tey can cause severe infections in individuals under specifc conditions [1].Any disruption in the host/commensal balance that weakens the host defense system or environmental factors, such as the use of antibiotics that inadvertently promote the growth of resistant enterococci, can lead to lifethreatening enterococcal infections [2].In recent decades, enterococci have become signifcant nosocomial pathogens and are frequently isolated from serious hospital-acquired infections, including infective endocarditis (IE); urinary tract infections (UTIs); bacteremia; nosocomial meningitis; and intraabdominal, wound, and pelvic infections [3].
Enterococci, although not typically highly virulent, pose a signifcant challenge in clinical settings due to their high resistance to antimicrobial agents [4].Te intrinsic resistance of these bacteria, along with their ability to acquire additional resistance mechanisms and to thrive in hospital environments, complicates the treatment of enterococcal infections [5].Te rise of high-level aminoglycoside resistance (HLAR) and vancomycin-resistant enterococci (VRE) in clinical isolates is a growing global concern, leading to more severe infections and posing serious health risks [6,7].Te increasing prevalence of HLAR and VRE isolates in areas such as Iran has alerted healthcare professionals, requiring strict infection control measures and presenting complex treatment challenges for clinicians.Actions to combat the spread of resistant enterococci strains are essential for maintaining public health and ensuring efective patient care [8][9][10][11].
Te prevalence and impact of drug-resistant enterococcal infections among pediatric patients remain inadequately understood, despite extensive research in adult populations.Tis knowledge gap underscores the importance of investigating the epidemiology and antibiotic resistance profles of enterococcal isolates in children.Te current study sought to address this gap by evaluating the prevalence and resistance patterns of enterococcal infections in pediatric populations for fve years.

Patients and Clinical Specimens.
Te present crosssectional study was carried out on children with any obvious enterococcal infection referred to the Children's Medical Center Hospital in Tehran from March 2016 to March 2021.Various clinical samples were collected from outpatients and inpatients (hospitalized patients).In our study, UTI was defned as an infection of the urinary tract with 10 4 CFU of cultured enterococci in urine and at least one UTI symptom, such as fever or urinary frequency.Only one isolate per patient was included in the study.Te patients' personal and clinical information was recorded, including age, sex, length of hospital stay, time and ward of strain isolation, and microbiological data.

Identifcation of Enterococcal
Isolates.Standard conventional biochemical tests for the identifcation of isolates were performed on colonies from the primary cultures.For this purpose, all suspected colonies of Enterococcus spp.were examined using Gram staining and further identifed using the catalase test, hemolysis examination, bile-esculin agar, and growth in 6.5% NaCl.Te isolates were stored at −70 °C in trypticase soy broth containing 10% glycerol for later analysis.

Detection of HLGR and VRE
Isolates.HLGR was detected using an agar-screening method.In brief, 10 μL of bacterial suspension adjusting to 0.5 McFarland turbidity standard was spotted on a brain-heart infusion (BHI) agar plate (HiMedia, India) containing 500 μg/mL gentamicin.Te plates were incubated at 35 ± 2 °C for 24 h.Te growth of more than one colony in the spotted zone was considered HLGR.Te control quality of the culture plates was determined by reincubation at 35 ± 2 °C for 24 h.VRE isolates were also detected by culturing on BHI agar supplemented with 6 μg/mL vancomycin (HiMedia, Mumbai, India), according to a previously described method [10,12].

Results
A total of 404 clinical isolates of Enterococcus spp.were obtained from children over fve years.Te patients' ages ranged from infancy (1 day) to 15 years.Te median patient age was 3 ± 1 months.47.5% of the patients were female, and 52.5% were male.
Te distribution of enterococci isolates based on the inpatient and outpatient clinics is outlined in Table 1.Te data reveal that 17.3% (70 out of 404) of the isolates originated from outpatients, whereas the vast majority, constituting 82.7% (334 out of 404), were sourced from inpatients.
In our study, all isolates were assessed for resistance rates without species diferentiation (Table 2), revealing high-level gentamicin resistance (HLGR) at 87.4% (90.9% in E. faecalis and 87.7% in E. faecium) and ciprofoxacin resistance at 98.2% (84.6% in E. faecalis and 99.3% in E. faecium).Ampicillin resistance rate was 60.9% (14.6% in E. faecalis and 78.8% in E. faecium), while vancomycin resistance was 49% (11.7% in E. faecalis and 63.5% in E. faecium) (Table 3).All the isolates were resistant to ampicillin-sulbactam. Te resistance rates of ampicillin, penicillin, and vancomycin in inpatients were twice as high as those in outpatients (Table 4).98% of the isolates were susceptible to linezolid and 2% were resistant (0% in E. faecalis and 2.2% in E. faecium).92.5% of the isolates were susceptible and 5.3% were resistant to nitrofurantoin (0% in E. faecalis and 7.9% in E. faecium).
Te antibiotic susceptibility results showed that linezolid was the most active antibiotic against the tested enterococcal isolates (98% susceptibility rate).Nitrofurantoin showed excellent activity against urinary enterococcal isolates (92.5% as the susceptibility rate).In addition, 49.3% (n � 199) of the isolates were identifed to have the VRE phenotype (12.6% in E. faecalis and 63.5% in E. faecium).As well as VRE phenotype was seen in 56.9% of inpatients and 12.9% of outpatients.Figure 1 shows a panel of VRE and non-VRE Enterococcus strains.
Forty-two point one percent (42.1%) of the total enterococci isolates were MDR (11.7% in E. faecalis and 54.2% in E. faecium).MDR enterococci isolates were detected in 48.8% of inpatients and 10% of outpatients.
Te HLGR phenotype was detected in 87.4% of E. faecalis, E. faecium, and other Enterococcus spp.isolates.Te majority (90.9%) of HLGR was found in E. faecalis isolates.Figure 2 displays a panel of HLGR and non-HLGR Enterococcus strains.
Altogether, fve of 404 (1.2%) enterococcal isolates had both VRE and HLGR phenotypes and were resistant to linezolid.In addition, resistance to linezolid and nitrofurantoin was only observed in E. faecium, and a greater number of E. faecium strains showed resistance to other antibiotics than E. faecalis strains.

Discussion
Since the late twentieth century, when enterococci have emerged as a signifcant cause of nosocomial infections, bacteria have become the second most common nosocomial pathogen and the third leading cause of nosocomial bloodstream infections [14].Enterococcal infections can include serious life-threatening disorders, especially in children, such as meningitis, septicemia, and infective endocarditis [15].However, few studies have been conducted on enterococci isolated from children in Iran.In our study, E. faecium and E. faecalis comprised 71.3 and 25.5% of the Enterococcus spp.isolated from pediatric patients, respectively.Tis observation is similar to reports from other regions of Iran and other countries, in which the distribution of enterococcal species derived from clinical samples, such as blood, urine, pleural fuid, cerebrospinal fuid, sputum, ascites, and hydrothorax, changed in favor of E. faecium [16][17][18][19].In contrast, some studies have shown that the frequency of E. faecalis isolates in clinical specimens is usually two to three times higher than that of E. faecium isolates [20,21].Tis diference may be due to the diferent origins of the collected samples and diferent geographical regions.Furthermore, the increase in the prevalence of E. faecium species may be due to the common resistance of enterococci to antienterococcal drugs such as ampicillin, aminoglycosides, and glycopeptides [22,23].In our study, the highest vancomycin resistance was found in E. faecium isolates.Te occurrence of VRE diverges in diferent countries, with a high frequency described in VRE in the US, the UK, Saudi Arabia, Ireland, and Turkey, whereas a low percentage is specifc to some European countries such as Italy and France [24,25].
Invasive enterococcal infections pose a signifcant challenge due to the emergence of drug-resistant strains.Te standard treatment of vancomycin, ampicillin, and aminoglycoside antibiotics is becoming less efective as resistance to these antibiotics grows [26].Vancomycin- Canadian Journal of Infectious Diseases and Medical Microbiology resistant Enterococcus (VRE) and high-level gentamicinresistant (HLGR) strains are particularly concerning, as they are responsible for a rising number of nosocomial infections in both adults and children [27].Identifying alternative treatment options for these drug-resistant enterococci is crucial for the efective management and treatment of such infections [28].Little data are available on the epidemiology and impact of VRE and HLGR infections in Iranian children.In this survey, we investigated the epidemiology and prevalence of clinical isolates of enterococci among the pediatric patients during the fve years.
Approximately one-third of the enterococcal strains were isolated from urology and PICU wards.Previously, a similar distribution was reported among hospitalized children in the urology ward located at the Children's Medical Center by Pourakbari et al. [29] and Sabouni et al. [30].Te high frequency of enterococcal strains isolated from the urology ward demands more attention to infection prevention and control (IPC) programs than in other units [31].
Enterococci are typically antibiotic-resistant. Owing to the intrinsic and acquired resistance to most antibiotics, the addition of vancomycin resistance meant that many To date, this is the largest high-risk pediatric population screened for VRE infections.A lower distribution of VRE (16%) was observed in another study conducted in children at the Children's Medical Center [29].In this study, E. faecium was the predominant VRE isolate (45.30%), which is consistent with the results of similar studies [34,35].In contrast, E. faecalis is the predominant VRE, which has been reported in other studies [36].Based on these fndings, a substantial increase in the hospitalization rate for VRE infection among pediatric patients has been observed over the last few years, paralleling the increase observed in the hospitalized adult population [37].Te reason for this increase in VRE incidence may be the amalgamation of the same factors, including selective pressure from broad-spectrum antimicrobial agents, an increasing number of critically ill and immunosuppressed at-risk patients, and increased nosocomial transmission owing to defciencies in hospital infection control practices [38].In addition, it has been reported that long-term use of vancomycin and cephalosporins can potentially lead to the selection of intestinal carriage of vancomycin-resistant enterococci (VRE) [39].It is important to remember that VRE infections are associated with high morbidity and mortality rates as well as increased duration and cost of hospitalization [40].Owing to the excellent performance of hospital infection control and accuracy in prescribing antibiotics in developed countries, a very low prevalence of VRE has been reported in countries such as Japan and the United States [37,41].Te majority of isolates showed more than 80 percent resistance to high levels of gentamicin in vitro.In numerous studies conducted both in Iran and other countries, a high prevalence of HLGR has been reported in E. faecalis [42,43].Tis may be attributed to its higher prevalence in clinical settings.However, our report deviates from this trend, as we observed a higher frequency of HLGR in E. faecium, aligning with the fndings of Hayakawa et al. [36].Tis divergence highlights the need for further research to understand the varying patterns of HLGR in diferent Enterococcus species.As well as, it is necessary to consider the origin of the clinic (inpatient or outpatient) when assessing resistance rates.Our study revealed that HLGR was signifcantly more common among inpatients compared to outpatients.Tis disparity in resistance rates among E. faecium isolates could potentially be attributed to this diference in clinical settings [19].
Overall, the isolation rate of HLGR was greater than that of VRE in our study, which is similar to the fndings of other studies [44].Te treatment of HLGR enterococci poses a signifcant challenge in healthcare settings.Recent reports have suggested the use of an antipeptidoglycan active agent in combination with a membrane-active agent to address this problem [45].Despite the predominance of HLGR enterococci in our region, vancomycin continues to demonstrate antimicrobial efcacy against such strains.However, the emergence of VRE isolates is a cause for concern and warrants continuous surveillance using both genetic and phenotypic methods to address this type of resistance.Tis "red alarm" highlights the importance of proactive measures in our health system to efectively manage and mitigate the impact of HLGR enterococci [46].
In our surveillance, a high prevalence of multidrugresistant (MDR) enterococcal isolates, defned as resistance to three or more antibiotic classes, was observed among pediatric patients.Te prevalence of MDR enterococci was more common in E. faecium (54.2%) compared to E. faecalis (11.7%), which is in concordance with previous studies [33,47].In addition, the MDR phenotypes were signifcantly higher for both E. faecium and E. faecium of inpatient isolates compared to those from outpatients.Canadian Journal of Infectious Diseases and Medical Microbiology It has been shown that the majority of VRE isolates had additional resistance to penicillin G, ampicillin, ampicillinsulbactam, and gentamicin.However, the resistance to these antibiotics was signifcantly lower than that in the non-VRE isolates.Generally, E. faecium is less susceptible to β-lactam agents than E. faecalis because their penicillin-binding proteins (PBPs) have lower afnities for these antibiotics [48].In addition, resistance mechanisms to other antibacterial agents, except β-lactams, could be encoded by conjugative transposons, and the presence of MDR isolates may facilitate the increase, transmission, and spread of drug resistance in hospital wards [49].Previously, clinical isolates of MDR enterococci have been frequently reported in several studies.In developing countries, unreasonable administration of antimicrobial agents, especially beta-lactam antibiotics, may be responsible for developing MDR enterococci [47].
Linezolid is used as the frst-line treatment for infections caused by VRE.Te potential coexistence of linezolid and vancomycin resistance among enterococcal strains also raises concerns, considering the importance of these drugs in the treatment of MDR enterococcal isolates [50].
In the present study, the rate of linezolid resistance was 2%, whereas various studies have reported 100% sensitivity to linezolid [51].An intriguing aspect of this study was the identifcation of four VRE strains from pediatric patients who were not susceptible to linezolid.Our results were similar to those of a recent study that found four clinical isolates of E. faecium that were resistant to both linezolid and vancomycin [52].
Limited treatment options are available for linezolid resistance VRE (LRVRE) with difcult-to-treat infections.Current treatment options such as linezolid, daptomycin, quinupristin/dalfopristin, and tigecycline have shown promising efcacy against VRE infections [53].Moreover, combination therapy may ofer a distinct advantage over monotherapies due to their broad spectrum and synergistic efect [54].Te use of quinupristin/dalfopristin (Q/D) in combination with rifampin or doxycycline and daptomycin and/or linezolid in combination with ampicillin, tigecycline, doxycycline, rifampin, and fuoroquinolones has been reported in some literature for the treatment of VRE infections [55].
Although molecular typing was not performed in this study to determine the potential clonality of the linezolidnonsusceptible VRE strain from the same region, this was a possibility, as the nosocomial spread of linezolid-and vancomycin-resistant enterococci has been previously described [56].
While linezolid nonsusceptibility remains sporadic among enterococci, constant surveillance of linezolid activity is required to control these opportunistic pathogens.

Conclusion
To our knowledge, this study is among the few to show the prevalence of VRE and linezolid nonsusceptible enterococcal isolates in pediatric patients.Terefore, efcient and accurate epidemiological typing methods such as pulsed-feld gel electrophoresis (PFGE) have been suggested for surveillance and to limit the occurrence and spread of epidemic clones within and between hospitals and community settings.Our study showed a rise in resistance to gentamicin and vancomycin, highlighting the need for a reassessment of antibiotic treatments in Iran.Moreover, to prevent the spread of VRE and linezolid nonsusceptible strains in healthcare settings, resistance measures derived from those used in many hospitals were implemented.Consequently, antimicrobial susceptibility testing is essential for managing enterococcal infections, and antibiotics should be prescribed carefully.Continuous monitoring of HLGR and VRE prevalence and drug susceptibilities for both inpatient and community isolates is highly recommended.

2. 5 .
Statistical Analysis.Data analysis was performed using Excel 2022 and IBM SPSS Statistics version 20.0 (Chicago, IL, USA).

Table 1 :
Distribution of enterococci isolates in inpatient and outpatient clinics.

Table 2 :
Antibiotic susceptibility testing of the enterococci isolates without species diferentiation.

Table 3 :
Antibiotic susceptibility testing of the enterococci isolates with species diferentiation.

Table 4 :
Antibiotic susceptibility testing of Enterococcus isolates according to their clinical source.
[33]isolates were obtained from inpatients, with a lower percentage originating from outpatients.However, the prevalence of VRE in a specifc population can vary signifcantly due to factors such as geographical location, duration of hospital exposure, usage of medical devices, sample size, and others[33].